Theeffect of strontium incorporated Zn-Ca-P coating on the biodegradability ofAZ31 alloy was evaluated. The Sr content and deposition time were optimized andcoated on AZ31 alloy by chemical conversion technique. The coating formed with1.
5 wt.% Sr and 20 min phosphating time at 50° C with pH 2.5 completely coversthe surface of the alloy. Sr doped coated sample showed evolved hydrogen volumeand pH value three times lower than Zn-Ca-P coatings which implied thecontrolled degradation of the coating. On immersion in Simulated Body Fluid,this surface exhibits high bioactivity with the deposition of calcium phosphatephases with Ca/P ratio of 1.
55 which is close to that of hydroxyapatite,mineral component of bone. Cytotoxicity evaluation with L969 cells showed thatSr doped coatings exhibited 72% cell viability on resorbable magnesium alloys.Keywords: Magnesium, Zinc Calcium Phosphate,biocompatible, corrosion, Strontium, cell viability. 1. IntroductionMagnesiumalloys have been evolving as a promising biodegradable material due to its favourablemechanical properties, biocompatibility and lightweight 1. Particularly, theelastic modulus of magnesium is closer to that of natural bone which avoidsstress shielding effect 2. Moreover, magnesium is an important element foundin human body and involved in body metabolic activities such as proteinsynthesis, muscle contraction and relaxation, energy transport etc.
3, 4. Lowlevels of magnesium lead to Alzheimer’s disease,asthma, attention deficit hyperactivity disorder (ADHD) 5. Even thoughmagnesium alloys have many favourable properties, electrochemical potential ofmagnesium is in the active region in the EMF series leading to rapid corrosion,increase in local pH, hydrogen evolution, loss of their mechanical integritybefore bone healing process 6, 7. The necessary requirement of biodegradableimplants is that the degradation rate should be matched with the healing rateof bone 8. For new bone formation, magnesium alloy should maintain itsmechanical integrity at least for 3 months. At present, magnesium alloys areused for biodegradable implant applications such as screws, pins andstents. Mainlythree approaches have been developed to control the degradation rate ofmagnesium such as alloying, surface modification and coating 9.
Coating isone of the key solutions to overcome the corrosion rate of magnesium alloy inchloride containing environment and provides some barrier effect between thematerial and the environment. Various surface treatment techniques such asphysical vapor deposition, electrodeposition, anodization, chemical vapourdeposition, microarc oxidation are available to achieve desired coating 10.Long treatment time, high cost, high temperature, complex procedures are thelimitations of these techniques. In contrast, conversion coating technique is asimple technique and can produce a highly adherent coating 11. Particularly,phosphate conversion coatings are regarded as suitable alternative compared toother conversion coating techniques to improve surface properties due to lowtoxicity 12. Zinc phosphate, calcium phosphate(CaP) and zinc calcium phosphate (Zn-Ca-P) coatings are attractive forbiomedical applications, as the elements present in the coatings are essentialfor human health 13-17. However, to be best of available literature, noattempt has been made to study the effect of ions in the Zn-Ca-P coating tofurther improve the bioactivity and corrosion resistance.Among various dopant ions, strontium is of our interest due to its biologicalperformance and bioactivity.
Hence Sr doped HA, bioactive glass, bone cementhave been synthesized 18-21. Sr doped CaP coatings have been investigated asan implant coating material 22. In view the advantages of both the coatingmaterial and coating technique, we have attempted the feasibility of Sr dopedZn-Ca-P coating by conversion coating method. By adding strontium precursor inthe phosphating bath, strontium incorporated PCC coatings can be developed.
Hence the present work deals with the optimization of strontium content in thezinc calcium phosphate conversion coating on magnesium AZ31 alloy and itsevaluation for bioactivity, corrosion resistance and cytocompatibility.2. Experimental Procedure2.1. Substrate preparationThesubstrate used in the present study is AZ31 magnesium alloy which was purchasedfrom Exclusive Magnesium, Hyderabad, India. The chemical composition of thesubstrate is 2.
9% Al, 0.88% Zn, 0.001% Fe, 0.
02% Mn, and balance magnesium. Thesubstrate was polished with SiC paper up to 1200 grit. The substrate was washedwith double distilled water and ultrasonically cleaned and degreased withacetone. Zn-Ca-Pand Sr doped Zn-Ca-P coatings were prepared on the surface of AZ31 alloy in thephosphating bath. The phosphating temperature was maintained at 50° C.
The pH ofthe bath was adjusted to 2.5 with the help of phosphoric acid. The compositionof the phosphating bath is 10g/L diammonium hydrogen phosphate ((NH4)2HPO4),7 g/L zinc nitrate (Zn (NO3)2), 3 g/L calcium nitrate(Ca(NO3)2), 3 g/L sodium nitrate (NaNO2), 1g/Lsodium fluoride (NaF). Sr doped zinc calcium phosphate coating was deposited onthe substrate by varying the strontium content (precursor used is strontiumnitrate) as 0.
5, 1 and 1.5 wt.% with optimized pH of 2.5 at 50° C (optimizedtemperature) for various deposition times i.
e. 5, 10, 15, 20 and 30 min. Zinccalcium phosphate coating was also deposited for comparison.2.
2 Surface characterization Fourier Transform Infrared (FTIR) spectra ofZn-Ca-P and Sr doped Zn-Ca-P coatings of various compositions were recorded onan FTIR spectrometer in the range of 400-4000 cm-1 with a singlereflection ATR accessory (Perkin Elmer Spectrum two, USA). Chemical compositionand phases of the compounds were analyzed using X-Ray powder diffractometer(XRD, D8 DISCOVER, Bruker, USA) using Cu k? radiation at 40kV and30mA at a scan rate of 0.02°. Scanning Electron Microscopy withEnergy-Dispersive X-ray spectroscopy (SEM, FEI, QUANTA 200, NETHERLANDS) wasused to characterize the surface morphology and elemental composition ofZn-Ca-P and Sr doped Zn-Ca-P coatings. Wettability of undoped and doped sampleswas measured using contact angle instrument (Easy Drop KRUSS, Germany) and SBFis used as contact liquid at different locations.2.3 Adhesion characterizationFilmswith good adhesion strength are considered as a protective overcoat and are veryvital for the protection against corrosion. Hence adhesion of the coating wastested as per ASTM (American Standards for Testing and Materials) D3359-09using tape adhesion test 23.
25 squares were developed by cross cutting thecoating in both directions using cross hatch cutter and adhesive tape wasapplied on the cross-cut area and pulled rapidly. Percentage of the adhesionremaining was calculated using the equation Adhesionremaining (AR) %= (n/25) × 100 ————————– Eq.1Evaluationwas made by substituting the number of peeled squares (n) in Eq.1 as per theASTM standard (0%-5B, <5%- 4B, 6-15%- 3B, 16-35%- 2B and 36-65%-1B). 2.4 In vitro degradation and mineralizationInvitro studies were conducted by immersing the samples in Simulated Body Fluid(SBF). The sufficient numbers of samples with equal dimensions were prepared bycoating process as explained in section 2.1.
The procedure for the preparationof SBF was reported by Kokubo et al 24. The uncoated and coated samples wereimmersed in the SBF kept at 37° C for 15 days. Hydrogen evolution test (HET)was carried out to study the evolved hydrogen from corroding specimen anddegradation of magnesium. The procedure for the HET experiment was inaccordance with the earlier report 23, 25. The initial pH of SBF wasmaintained as 7.2 and pH were noted down at different time intervals. Corrosioncurrent density obtained from an electrochemical method is easy and corrosionbehavior can also be monitored with time.
However, due to the specialelectrochemical behavior of magnesium called negative difference effect, thepolarization curve will be distorted and the reaction mechanism aroundcorrosion potential is different from that of tafel region. One hydrogen gasmolecule is generated by dissolving one atom of magnesium and corrosion rate isdirectly reflected by hydrogen evolution rate. Hence corrosion rate fromhydrogen evolution test is reliable. Corrosion rate of the samples wascalculated using the following equation, Corrosion rate (CR) = ——————-Eq.2 WhereCR is the corrosion rate in mg/cm2/h, ‘A’ is the initial surfacearea in cm2, ‘t’ is the immersion time and ? is the density of thesample (1.74g/cm2). ?g is themultiplication factor for calculating the amount of magnesium from the evolvedhydrogen volume as 1mL of H2 is given by dissolving 0.
001083 andthis value is used for the calculation.2.5 MTT AssayL969cells (from NCCS, Pune) were cultured with Dulbecco’s modified Eagle’s mediumsupplemented with 10% fetal bovine serum, 1% 100× antibiotic antimycotic liquidand incubated in a CO2 incubator at 37° C. A cell suspension with DMEMcontaining 1×105 cells were seeded on the sample which was placed inthe 9 well cell culture plate and MTT assay was conducted by incubating for 72h at CO2 incubator. 200 µL of cell culture medium was added to eachwell. Then 20 µL MTT solution was added followed with incubation for 4 h at37°C. The amount of reduced formazan product shows the number of viable cells.
Plate reader was used for the quantification of formazan by absorbance at 570nm. All the experiments were performed triplicates.3. Results and Discussion3.1 FTIR AnalysisFTIRspectra of Zn-Ca-P and Sr doped Zn-Ca-P coated samples formed with differentstrontium nitrate contents are shown in Fig.
1. The phosphate coating shows twostrong absorption bands between 900 and 1150 cm-1 which is due tothe stretching vibration of PO43- group. Thecharacteristic bending vibrations of phosphate groups are formed at 550, 610and 730 cm-1 26.Thebroad band around 3250 cm-1 and small band around 1680 cm-1correspond to OH- vibration 27.
Although the spectra look similarto each other, increase in the peak intensity of PO43-vibrations around 1026 cm-1 with increasing strontium content in ZCPcoating has been observed. The high intense peak of PO43-vibrations has appeared for 1.5 wt.% Sr doped Zn-Ca-P coating. 3.2 XRD AnalysisFig.
2shows the XRD patterns of pure and Sr doped Zn-Ca-P coated samples with variousSr(NO3)2 contents. All the XRD pattern closely resemblewith each other. The main diffraction peak of (3 2 1) of zinc calcium phosphate(JCPDS 98-000-427) and (2 4 1) of zincphosphate (JCPDS 98-001-8145) and (0 4 0)of strontium phosphate (JCPDS 96-153-3308) areclearly shown in Fig.2. It is evident from the XRD analysis that the zinccalcium phosphate coating contains zinc phosphate and zinc calcium phosphatephases.
In addition, strontium phosphate phase is also appeared in Sr dopedZn-Ca-P coated samples 28.3.3Coating mechanism Chemicaletching of phosphoric acid with magnesium produces a chemically favourablesurface for the formation of the subsequent coating. Hydrolyzation ofphosphoric acid derives H+ ions which in turn react with magnesiumions. Remaining Mg2+ ions react with HPO42-and produce strong adhesive phosphate layer Mg3(PO4)2)on the substrate which makes the medium more alkaline.Thealkalization of the solution facilitates the precipitation of insolublephosphates such as Zn2Ca(PO4)2, Zn3(PO4)2and Sr3(PO4)2.
The coating methodology wasschematically illustrated in Fig.3. The deposition process contains followingsteps a) dissolution of magnesium b) local increase in pH c) precipitation ofmagnesium phosphate d) formation of alkaline pH surrounding the implant e)precipitation of Zn2Ca(PO4)2, Zn3(PO4)2and Sr3(PO4)2.
From XRD results, Zn3(PO4)2 andZn2Ca(PO4)2 formation in the undoped coatingand additional Sr3(PO4)2 formation in thedoped coating is confirmed. Temperature, pH, treatment time mainly contributein the formation of compact two layers. 3.4 SEM AnalysisFig.4shows the surface morphologies of phosphate coatings at different phosphatingtime. Fig.
4 (a, b, c, d and e) shows the surface morphology of the samplesafter immersion in the Zn-Ca-P phosphating bath for 5, 10, 15, 20 and 30 minrespectively. Few white deposits were randomly deposited on the surface duringthe initial period.Asthe phosphating time was increased, the size of the deposits became larger andcovered the surface. The maximum surface coverage was found at 20 mindeposition time.
Some cracks were found in the coating due to internal stressesand also due to the evolved hydrogen from the dissolution of the magnesium ofthe alloy matrix. Morphology of Zn-Ca-P coating was totally different from thatof Sr-doped Zn-Ca-P coating which consists of needle like morphology. Fig.4 (f,g, h, i, and j) shows the surface morphology of Sr doped Zn-Ca-P coatedsamples. The randomly scattered particles formed on Sr doped Zn-Ca-P coating atinitial deposition time could not effectively protect the surface from SBF.
Incontrast, the coating formed at 20 min deposition time covered the entiresurface, though some defective regions were visible. Normally there was ageneration of hydrogen from the corroding magnesium due to the micro galvaniccorrosion between the particle and its ?-magnesium matrix 29. But small voidsin the coating offers pathways for the hydrogen and thus damage in the coatingis very much reduced. Moreover, Sr doped samples described herein controlledthe magnesium degradation greatly than other conversion coated samples. Maximum surface coverage and presence ofbioactive strontium phosphate phase combinedly seems to influence in protectingthe magnesium degradation in SBF. The maximum surface coverage also minimizedthe diffusion of SBF through the coating.
Strontium phosphate present in thecoating has a low solubility product value (1× 10-31), whichdissolves very slowly when implanted in the human environment 30. When thedeposition time was extended up to 30 min, some of the crystal deposits weredissolved in the phosphating bath and the surface was exposed again (Fig. (e)and (j)).
Hence 20 min deposition time is fixed as optimum deposition time andZn-Ca-P coating with varying Sr(NO3)2 content was studiedand the morphology of the coatings are shown in Fig.5. Low strontium nitratecontent in Zn-Ca-P coating did not cover the entire surface of the alloy. Asthe strontium nitrate content was increased to 1.
5 wt.%, the surface wasentirely covered with slab like crystals. Insert shows the compactly arrangedslab like crystal morphology. The coating formed at 50°C was more compactcompared to low phosphating temperature.
If coating temperature is furtherincreased, the coating dissolves in the acidic medium. Hence 20 min deposition time,50° C temperature and 1.5 wt.% strontium content seems to be optimal parametersfor the formation of a crack free coating. 3.
5 Contact angle measurementsThecontact angle values of uncoated, Zn-Ca-P and Sr doped Zn-Ca-P coatedsubstrates are shown in Fig.6. The contact angle of the uncoated alloy is foundto be 89°, whereas, for Zn-Ca-P and Sr dopedZn-Ca-P coatings, the values are 97° and 112° respectively. High contact anglevalues suggest that the surface energy of coated surfaces is low resulting inthe hydrophobic nature of the sample. Hence, when a drop is placed on thesurface, it does not spread on the surface and exhibits corrosion resistantbehavior.3.6 In vitro biomineralizationTheSEM-EDX analysis of AZ31, Zn-Ca-P and Sr doped Zn-Ca-P coated samples duringthe immersion in SBF for 15 days are shown in Fig.
7. The surface of theuncoated AZ31 alloy was severely cracked. The deposited calcium phosphatepeeled off from the surface due to the continuous corrosion reaction and henceless amount of calcium depositions can be seen on AZ31 after 15 days ofimmersion (Fig.7c). Incase of Zn-Ca-P coating, stable deposits were observed on the surface and sizeof deposits were found to increase from 38 µm to 43 µm during the immersiontime.
However, Sr doped Zn-Ca-P coating exhibits the presence of deposits onthe 5th day of immersion itself and the entire surface was coveredwith deposits of larger diameter (54 µm) (Fig.7i) at the final immersionperiod. The strontium present in the coating seems to assist in the depositionof more calcium from SBF and the calcium, in turn, withdraws more phosphatefrom the solution. Hence calcium phosphate with larger diameter was depositedin case of Sr doped Zn-Ca-P coated samples.
EDXspectra of AZ31, Zn-Ca-P and Sr doped Zn-Ca-P, coated samples after 15 days ofimmersion are shown in Fig.7 (j, k, l). All the spectra confirm the presence ofMg, Al, Zn, Ca, and P elements. The Mg, Al, and Zn elements are from thesubstrate and Ca and P elements are from SBF.
The Ca/P ratio calculated fromEDX results for AZ31 was 0.67, while the ratio of Zn-Ca-P and Sr doped Zn-Ca-Pcoating were 1.26 and 1.
55 respectively. The highest Ca/P ratio as close tothat of hydroxyapatite (Ca/P = 1.67) obtained for Sr doped alloy indicated theimproved biomineralization and hence protection from corrosion. 3.7 Adhesion characterizationAdhesionof the coatings was tested by cross cut adhesion test. The coating was presentin 25 squares (n=0) after the test for both coatings. Hence the value was substituted in eq.
1 andAR % calculated was found to be 0% and it falls under the highest grade 5B.Both the coatings seem to have good adhesion property without much delaminationof the coating.3.8 Hydrogen gas evolutionThehydrogen evolution results of the uncoated, Zn-Ca-P and Sr doped Zn-Ca-P coatedsamples in SBF are shown in Fig.
8. The total evolved volume of hydrogen gas ofAZ31 is 4.5 mL. In contrast, hydrogen evolution rate of all the coated samplesshows significantly lower values than AZ31 that could be tolerated by the humanbody. This may be due to the difficulty in charge transfer from solution to thealloy surface leading to good corrosion protection of the coated samples.Thedecrease in hydrogen evolution rates of Zn-Ca-P and Sr doped Zn-Ca-P coatingmay also due to high adhesion strength of the coating. Moreover, the depositedcorrosion products from SBF also seem to help in delaying the degradationprocess.
Particularly 1.5 wt.% Sr doped Zn-Ca-P coating has lowest hydrogenevolution due to the thick chemically stable and low soluble protective layer. 3.9 pH MeasurementsThepH variation of SBF solution during the immersion of uncoated and coatedsamples for 96 h are shown in Fig. 9. The pH values of SBF during the immersionof coated samples are lower than that of the uncoated alloys over the entire immersiontime. The low pH increase of Zn-Ca-P coating than AZ31 is due to the presenceof corrosion resistant and chemically stable zinc phosphate and zinc calciumphosphate in the coating.
Sr doped Zn-Ca-P samples showed lowest pH increase.This may be due to the presence of strontium phosphate which has low solubilityproduct value. Once it is coated on the surface, it will not reprecipitate fromthe surface and deposits in the phosphating bath. Also, strontium draws extracalcium and phosphate from the phosphating bath leading to the formation of thethick and compact coating. 4.
0 Corrosion rate calculation fromHydrogen Evolution TestThecorrosion rate of AZ31 by conventional electrochemical methods may not bereliable due to negative difference effect of magnesium and hence the corrosionrate was calculated from hydrogen evolution studies using the Eq.2 and it isshown in Fig 10. The corrosion rate of AZ31, Zn-Ca-P, and 1.5 wt.
% Sr dopedZn-Ca-P coated sample at the end of 96 hr is 2.42, 0.84 and 0.67 mg/cm2/hrespectively.
It is remarkable that the corrosion rate of Sr doped Zn-Ca-Pcoated sample is one fourth of AZ31 during the initial immersion time.4.1 MTT AssayThecell viability of L969 cells on AZ31, Zn-Ca-P,and Sr doped Zn-Ca-P coated samples by MTT assay are shown in Fig.11. All thesamples were found to be nontoxic with 60% cell viability at the end of 72 h.More viable cells were present in Zn-Ca-P and Sr doped Zn-Ca-P coated samplesthan that of AZ31.
Sr doped Zn-Ca-P coated sample showed the highest number ofviable cells compared to other samples which may be due to the chemicalstability of the coating.5. ConclusionsOptimalparameters such as pH, temperature, phosphating time and strontium content forthe deposition of Sr doped Zn-Ca-P coating using chemical conversion techniquewas obtained. Sr doped Zn-Ca-P coated sample exhibits low pH, evolved hydrogenvolume and corrosion rate. Strontium phosphate does not easily dissolve in theSBF solution due to its low solubility product value which provides moreprotection in the coating. This coating showed improved bioactivity as depositshaving Ca/P ratio closer to hydroxyapatite due to the higher induction ofcalcium and phosphorous by strontium from SBF solution. Hence Sr doped Zn-Ca-Pcoated AZ31 alloy may be a suitable degradable implant for orthopedicapplications. AcknowledgementOneof the authors, Dr.
P. Amaravathy thanks, Department of Science andTechnology-Science and Engineering Research Board (DST-SERB), Government ofIndia for providing fellowship under National Post-Doctoral Fellowship (NPDF)scheme.